Hepatitis C virus (HCV) infection is a major health problem in both developed and developing countries. It is estimated that about 1 to 5% of the world population is affected by the virus. HCV infection appears to be the most important cause of transfusion-associated hepatitis and frequently progresses to chronic liver damage. Moreover, there is evidence implicating HCV in induction of hepatocellular carcinoma. Consequently, the demand for reliable diagnostic methods and effective therapeutic agents is high. Also sensitive and specific screening methods of HCV-contaminated blood-products and improved methods to culture HCV are needed.
HCV is a positive stranded RNA virus of approximately 9,400 bases which encode at least three structural and six non-structural proteins. Based on sequence homology, the structural proteins have been functionally assigned as one single core protein and two envelope proteins: E1 and E2. The E1 protein consists of 192 amino acids and contains 5 to 6 N-glycosylation sites, depending on the HCV genotype. The E2 protein consists of 363 to 370 amino acids containing 9 to 11 N-glycosylation sites, depending on the HCV genotype (for review see Major and Feinstone, 1997; Maertens and Stuyver, 1997). The E1 protein contains various variable domains, while the E2 protein contains two hypervariable domains, of which the major domain is located at the N-terminus of the protein (Maertens and Stuyver, 1997). The envelope proteins have been produced by recombinant techniques in Escherichia coli, insect cells, yeast cells and mammalian cells. The usage of an expression system in higher eukaryotes and especially in mammalian cell culture leads to envelope proteins of superior quality, i.e. they are effectively recognised by antibodies in patient samples as described in PCT/EP 95/03031 to Maertens et al.
Currently, the detection of HCV in cells or tissues relies mainly on the demonstration of viral RNA. However, RNA detection in cells or tissues is a cumbersome technique which either involves the extraction of RNA followed by reverse transcription and nested PCR or includes in situ RT-PCR and hybridisation. Since these techniques are also prone to false positive reactivity, viral RNA detection is exclusively performed on serum samples. Reliable methods for the detection of viral protein antigens in serum and tissue samples are still lacking.
The replication sites of HCV have not yet been fully elucidated. It is generally accepted that the virus replicates in hepatocytes, but replication in other tissues, such as lymphoid tissues, is still highly debated. Hence, a reliable method for the detection of viral proteins or the virus itself in cells may solve this issue.
The detection of viral proteins in cells has been hampered by the lack of antibodies which specifically bind to viral proteins and which are able to specifically recognise the natural HCV protein antigens (i.e. antigens as expressed by the host following infection with HCV). As a consequence, to date only few studies relate to demonstrating the presence of viral HCV proteins in host cells (for review see Guido and Thung, 1996). Moreover, host-derived antibodies have been used in many of these studies. Preparations containing host-derived antibodies, however, cannot be reproduced easily and these preparations may be contaminated by autoimmune antibodies as well as by antibodies against other known or even unknown agents. In contrast, it is known that antibodies produced in animals upon immunisation with recombinant antigens will yield antibodies with the desired specificity. In order to have reproducible quality, monoclonal antibodies are preferred as well. In addition, the envelope proteins of HCV need to be produced by a mammalian expression system to yield good quality antigens. Currently, this expression condition goes together with incomprehensible problems. Therefore, only few monoclonal antibodies have been described which could be used to detect HCV envelope antigens in tissue specimens of patients. These antibodies were directed against the N-terminal region of E1, amino acids (aa) 192-226, (Hiramatsu et al., 1992, Kaito et al., 1994) or the C-terminal domain of E2, aa 451-715 (Sansonno et al., 1997a, 1997b). However, from these publications and from the reviews by Guido and Thung (1996), and Liang (1996) it is evident that there is still an existing need for well-characterised antibodies allowing efficient and routine detection of natural HCV protein antigens in serum and tissue samples. This need was recently confirmed by a study by Dries and co-workers (1999). Dries' group proved that 61% of HCV antibody positive and serum RNA negative individuals were carriers of HCV as HCV RNA could be detected in liver biopsy samples but only one third of these cases could be detected by immunohistochemistry using a panel of antibodies. Thus, routine detection of HCV antigen in liver biopsies still lacks sensitivity. Also the detection of viral antigens in body fluids such as serum or plasma is hampered by the same problem. Up to date only one single technique has been described allowing detection of core antigen in these fluids. However, in order to detect the core protein, the technique requires a complete denaturation of the core protein present in the sample using sodium hydroxide (Kashiwakuma et al., 1996).
Taken together, the identification of new specific HCV epitopes which are accessible for antibodies and which allow antigen detection in tissue or body fluid samples is therefore urgently needed. The HCV envelope proteins are putative candidate targets to find such epitopes, since these proteins should be present in all biological samples: in fluids, on the membrane of the virus, and in cells from the earliest event of infection (i.e. viral entry) throughout the complete replication cycle. However, these envelope proteins are highly variable so antibodies with a high cross-reactivity towards the different genotypes of HCV are needed. Thus, identification of such epitopes and the search for antibodies with high cross-reactivity towards the sequence variation of HCV is a challenging undertaking.
The present application relates to specific monoclonal antibodies, directed against particular epitopes in the envelope proteins of HCV, which are able to detect HCV antigen in tissue specimens of patients. In total two such epitopes, and corresponding antibodies, were found: one in the C-terminal region (aa 227-383) of the envelope protein E1 and one in the N-terminal hypervariable region (HVR) of E2 (aa 384-450). Although the latter region, and more specifically the region 395-415, is considered to be hypervariable, we characterised, to our surprise, an antibody which reacts with various known sequences of the HVR of E2.